S B 

741 

Pg J (a VITED STATES DEPARTMENT OP AGRICULTURE 

BULLETIN No. 759 

Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR, Chief 



Washington, D. C. 



PROFESSIONAL PAPER 



June 19, 1919 



THE LEAF-SPOT DISEASES OF ALFALFA AND 

RED CLOVER CAUSED BY THE FUNGI PSEU- 

DOPEZIZA MEDICAGINIS AND PSEUDOPEZIZA 

TRIFOLII, RESPECTIVELY 



By 



FRED REUEL JONES, Pathologist, Cotton, Truck, and 
Forage Crop Disease Investigations 



CONTENTS 



Scope of the Investigation 

The Diseases 9 

Economic Importance 2 

Description of the Disease on Alfalfa 3 
Description of the Disease on Red 

Clover j. ^ 

Host Plants 5 

The Fungi 5 

Synonomy of Pseudopeziza trifolii . 5 
Synonomy of Pseudopeziza medi- 

caginis P 

Comparative Morphology of the Fungi 6 

Morphological Characters in Culture 8 



The Fungi— Continued. 

Reported Conidiai Stages of \ These 

Fungi g 

Physiology of the Fungi 1 1 

Pathogenicity of the Fungi .... 19 
Life History of the Causal Organism in 

Relation to the Host Plants .... 27 
American Studies Bearing on Life 

History 27 

Method of Overwintering .... 28 

Method of Distribution 30 

Summary ,35 

Literature Cited 35 




WASHINGTON 
GOVERNMENT PRINTING OFFICE 

1919 






^^ 



■■•■•. •- 




Class. 

Book vlL ^ J (o 



<##* 



UNITED STATES DEPARTMENT OF AGRICULTURE 

BULLETIN No. 759 




Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR, Chief 




Washington, D. C. 



PROFESSIONAL PAPER 



June 19, 1919 



THE LEAF-SPOT DISEASES OF ALFALFA AND RED 
CLOVER CAUSED BY THE FUNGI PSEUDO- 
PEZIZA MEDICAGINIS AND PSUEDOPEZIZA TRI- 
FOLII, RESPECTIVELY. 

By Fred Retjel Jones. Pathologist, 
Cotton, Truck, and Forage Crop Disease Investigations. 



CONTENTS. 



Page. 

Scope of the investigation 1 

The diseases 2 

Economic importance 2 

Description of t he disease on alfalfa 3 

Description of the disease on red clover . . 4 

Host plants 5 

The fungi 5 

Synonomy of Pseudopeziza trifolii 5 

Synonomy of Pseudopeziza medicaginis. 6 

Comparative morphology of the fungi — 6 

Morphological characters in culture 8 



Page. 



The fungi— Continued. 

Reported conidial stages of these fungi . . 9 

Physiology of the fungi 11 

Pathogenicity of the fungi 19 

Life history of the causal organism in relation 

to the host plants 27 

American studies bearing on life history. 27 

Method of overwintering 28 

Method of distribution 30 

Summary 35 

Literature cited 36 



SCOPE OF THE INVESTIGATION. 

Among the diseases of the foliage of the alfalfa plant, the one 
which is most widely known and is reported to cause the greatest loss 
is the leaf-spot caused by the fungus Pseudopeziza medicaginis. The 
disease is commonly called the alfalfa leaf -spot. This name is not 
distinctive, and its continued use is open to the objection that it 
promotes the present tendency to apply it inclusively to all the sev- 
eral leaf diseases that may be present with the true leaf -spot. How- 
ever, the usage is so prevalent that it appears likely to persist. 

Although the importance of the disease has caused it to be men- 
tioned widely and frequently, little careful study has been devoted 
to it. A great number of scattered and conflicting observations have 
kft the life cycle of the fungus causing the disease in doubt; the 
89950°— 19 1 



2 BULLETIN 759, TJ. S. DEPARTMENT OF AGRICULTURE. 

method of overwintering of the fungus has not been conclusively 
demonstrated; and the oft-considered question whether this fungus 
is identical with or merely similar to one which causes a correspond- 
ing leaf-spot on red clover has never been decisively answered. It 
is because of the last consideration that the two diseases have been 
studied together. The leaf -spot of red clover caused by the fungus 
Pseudopeziza trifolii occurs over a wide range of territory, but 
usually not so abundantly as that on alfalfa. Mention of it occurs 
frequently in literature. No distinctive common name has been ap- 
plied to it. Much of the later interest in this disease is in connection 
with the question whether or not red clover is a source of infection, 
producing destructive outbreaks of leaf-spot on neighboring alfalfa 
when this plant has been introduced into new localities. 

Leaf-spots of a nature very similar to the two already mentioned 
and commonly reported to be caused by the same fungi are known 
on a long list of clovers, alfalfas, and closely related plants. All of 
these diseases should be studied together and the relationship of the 
causal organisms determined. However, most of them do not occur 
in America or only in restricted localities, and none of them causes 
great damage to the host plants. The only one of these of which any 
living material has been available for study is that caused by 
Pseudopeziza medicagmis on Medicago lupullna. The incomplete 
notes on this disease have been included. 

THE DISEASES. 

ECONOMIC IMPORTANCE. 

As has already been stated, the assertion has been made again and 
again that leaf-spot is the most common and destructive of the 
foliage diseases of alfalfa. That it is the most common is beyond 
question. But in the estimates of the loss which it has caused it 
appears highly probable that damage from other causes than the 
conspicuous leaf -spot has been included. Nevertheless, even if proper 
deductions for these inclusions could be made it might still be true 
that leaf -spot causes greater loss than any other foliage disease. 

The highest estimate of loss from this disease is that of Pammel 
(1891) x from Iowa. In 1890 he attributes to this cause a loss of 
half the crop. Stewart and others (1908, pp. 384-387) report from 
New York that young stands are often ruined and that old stands 
are killed outright. Chester (1891) reports that some plats at the 
Delaware station in 1889 were attacked severely before the plants 
were large, and some of them were completely destroyed. Voges 
(1909) in Germany and Ivy Massee (1914) in England note the 
sickly appearance of diseased fields. 

1 The dates in parentheses refer to " Literature cited " at the end of this bulletin. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 6 

The amount of damage which the fungus may cause appears to de- 
pend on several circumstances relating to the development of the 
crop and the weather. Under ordinary conditions the incubation 
period of the disease is more than a week. If for any reason the 
plant is growing slowly, the stand is thick, and the weather is fre- 
quently wet, only a few of the upper leaves reach full development 
before they are covered with the disease. Thus young stands which 
grow slowly before becoming firmly rooted and old stands which are 
retarded for any reason are likely to show bad attacks, while stands 
which are growing rapidly keep most of the upper leaves well above 
the rising invasion of the fungus and show little harm. Thus, in 
most cases where the fungus is found in great abundance, apparently 
defoliating plants, it will be found that some condition has reduced 
the normal rate of growth of the plants and is in part responsible 
for the resulting damage. When plants are vigorous, infection must 
be heavy indeed to cause extensive yellowing and falling of leaves, 
though this may occasionally occur. 

Nevertheless, the fungus is present in almost every alfalfa field, 
if not in all fields, taking a small toll of the foliage under even dry 
conditions and a large toll under more humid conditions. Since it 
rarely produces great loss at one time it has come to be regarded as 
one of the unavoidable evils to which the alfalfa plant is subject. 

The leaf-spot of red clover caused by the fungus Pseudopeziza 
irifolii has not frequently been reported as occasioning great loss. In 
Russia Jaczewski (1912, p. 98) speaks of it as causing appreciable 
damage. Blasdale (1902, p. 75) states that it injures nearly all the 
clovers of the stock ranges of northwestern California. Freeman 
(1905, p. 309-310) notes that it causes local epidemics in clover fields 
in Minnesota. In fields in northern "Wisconsin and in Maine in the 
summer of 1915 it was observed by the writer to be so abundant as to 
cause appreciable loss of foliage. From the evidence at hand it 
appears that the disease is not of great significance to clover and 
that this significance is only in northern regions. However, the 
destroyed foliage is so much less conspicuous than that on alfalfa 
that the amount of damage is more likely to be underestimated than 
overestimated. 

DESCRIPTION OF THE DISEASE ON ALFALFA. 

There are two characteristics of the leaf-spot caused by Pseudo- 
jieziza medlcaginis which usually serve to distinguish it from spots 
caused by other parasitic fungi. The first of these is the circular 
shape and limited size of the spot. (PL I, .4.) The second is the 
presence of a small raised disk (PI. II, B) that appears in the center 
of the spot when it has reached full development. The edge of the 
spot may be smooth and definite, especially if the leaf has been much 



4 BULLETIN" 759, U. S. DEPARTMENT OF AGRICULTURE. 

exposed to the sun, or it may be more or less dendritic, with a fringe 
of olive-colored rays. No marked killing or sinking of the leaf 
tissue occurs. In size, the spot rarely exceeds 2 or 3 millimeters in 
diameter. 

The disk at the center of the spot is the fruiting structure of 
the fungus and consists of a mass of asci which discharge large 
numbers of spores when sufficient moisture is present. These disks 
usually occur on the upper side of the leaf, sometimes on the lower 
side, and rarely on both sides from the same spot. Typically they 
are 1 to 1^ millimeters in diameter, slightly raised, and when fully 
developed surrounded by the torn edges of the epidermis of the leaf. 
Rarely the central disk is found surrounded with several smaller 
disks at its margin. Under very moist conditions the disk may ap- 
pear as a'jellylike drop of exudate at the center of the spot. Under 
arid conditions it becomes very dark in color, often almost black. 

There is not usually a striking difference in color between the dis- 
eased tissue and the disk at the center. This color varies from dark 
brown to almost black. If the leaf has begun to yellow, the green 
color is sometimes retained longest around the diseased area. 

The disease often occurs on succulent stems, where it has an ap- 
pearance so characteristic that it can hardly be confused with injury 
from any other cause. The spot is elliptical in shape, with perfectly 
smooth edges. In size it is about 1^ by 3 mm. It is not abundant, 
and rarely bears a fruiting disk. 

DESCRIPTION OF THE DISEASE ON RED CLOVER. 

The leaf-spot on red clover caused by Pseudopeziza bears a very 
close resemblance to the similar disease on alfalfa. The spots are 
limited in size, usually slightly larger than on alfalfa, in early 
stages tending to be angular (PI. I, C). The border of the spot is 
more frequently dendritic in outline. In early stages the color is 
dark olive, becoming brown or almost black in later stages. After 
the death of the entire leaf, the spot usually becomes almost indis- 
tinguishable. Fruiting disks are not as frequently found abundantly 
on the spots while the leaf is still alive as in the case of the leaf-spot 
of alfalfa, but they may develop abundantly after the death of the 
leaf. In early stages they are brownish or dirty yellow, but later 
they become almost black. They are more frequently found on the 
under side of the leaf than on the upper side, and occasionally occur 
on both sides from the same spot. On dead leaves they appear as 
amber drops of jelly in wet weather, but when dried they shrink to 
bodies so small and inconspicuous that it is practically impossible 
to find them. The disease has not been noted on any other part of 
the plant than the leaves. 



Bui. 759, U. S. Dept. of Agriculture. 



Plate I. 







*- 



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-& # 



PSEUDOPEZIZA ON HOST PLANTS AND PURE CULTURES. 

A, Leaf-spot of Mcdkago lupulina caused by Pseudopeziza mcdkaginis (Lib.) Sacc. B, Pse udopeziza 
medicaginis developing in pure culture from spores discharged on plaster of Paris (right) and niter 
paper (left). C, Leaf-spot of red clover caused by Pseudopeziza trifohi (Biv.-Bern.) Fckl. at an early 
stage of development. Apothecia have not yet appeared. 



Bui. 759, U. S. Dept. of Agriculture. 



Plate 1 1 . 



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4 ' * 



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PSEUDOPEZIZA ON ALFALFA. 

A, Apothecia of Pseudopoziza developing on old leaf-spots on overwintered leaves. In many cases 
the apothccia are clustered. X 3, approximately. B, Apothccia of Pseudopoziza on an alfalfa 
leaiict. the leaf has been decolorized to show the fungus more clearly. The small dark circles 
near the lower right-hand edge of the leaf are early stages of apothccia of Pyrenoncziza mcdicaqinis. 
X 5, approximately. » r v 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 5 

HOST PLANTS. 

Although these fungi occur extensively in America on alfalfa and 
clover only, each of them has been reported in mycological literature 
on a number of related plants. 

The names of these host plants are shown in Table I. 1 

Table I. — List of -host plants of Pseudopeziza trifolii and Pseudopeziza 

medicaginis. 



Hosts of Pseudopeziza trifolii. 


Hosts of Pseudop 


iziza medicaginis. 


Name of host plant. 


Authority cited. 


Name of host plant. 


Authority cited. 


Trifolium: 


Saceardo, D., 1903,no.526, 

1319. 
Krieger, 1892, no. 781, 794. 
Blasdale, 1902. 
Massee, 1914. 
Bivona- Bernardi, 1S16, 

man. 4, p. 27, pi. 6, fig. 5. 
Saceardo, P. A., 1889, pp. 

723-724. 
Lambotte. 18S0, p. 264. 
Blasdale, 1902, p. 75. 

Briosi, 1888. 
Massee, 1914. 
Saceardo, P. A., 1889, 

pp. 723-724. 
Maire, 1913, no. 119. 
Briosi, 1888. 
Massee, 1914. 
Saceardo, P. A., 1897, p. 

623. 


Medicago: 

carstiensis Jacq 


Saceardo, T>., 1903, no. 




526, 1319. 
Cavara, 1892, p. 243. 


cvathiferum Lindl . . 

ff agiferum L 

hybridum L 

incarnatum L 


hispida denticulata 
(Wild.) Urban. 


Massee, 1914. 


lupulina wildenowii 
(Boenn.) Aschers. 

minima Link 

prostrata Jacq 


Libert, 1832, fasc. 2, no. 

176. 
Fuckel, 1S70, p. 290, 236. 
Jaap, 1916. 


macrodon Hook and 
Am. 

nigrescens Viv 

pallescens Schreb 


Desmazieres, 1847, pp. 


Melilotus alba Desv 

Onobrychis sativa Link . 
Trigonella: 

coerulea (L.) Ser 

corniculata L 

foenum-graecum L . . 
Vicia villosa Roth 


182-183. 
Tracy and Earle, 1895, 

p. 106. 
Berthault. 1913. 


pallidum W. and E. 


Mazerius, 1S75, fasc. 33, 
no. 1645. 


spadieeum L 


Jaap, 1916. 
Massee, 1914. 
Tracv and Earle, 1895, 
p. 106. 



THE FUNGI. 

SYNONOMY OF PSEUDOPEZIZA TRIFOLII. 

Pseudopeziza trifolii was first described by Antonio Bivona- 
Bernardi (1816, Mar. 4, p. 27, pi. 6. fig. 5) on Trifolium hybridum 
from Sicily under the name of Ascobolus trifolii. When Boudier 
(18G9) revised the genus Ascobolus he listed this species among 
those which he believed should be excluded, and suggested that it 
be placed in the genus Phacidium. The following year Fuckel 
(1870) made this species the type of his new genus Pseudopeziza. 

Other early synonyms as given by Eehm (1892, p. 597-598) are 
as follows: Peziza trifpliorum Libert, Trochilia trifolii DeXot., 
Molliscia trifolii Phill., Phyllachora trifolii Sacc. 

The following three names have been included in the synonomy 
by Ivy Massee: Pseudopeziza (Phacidium) divergens (Desmaz.) 
Sacc. ; Peziza dehnii Rab., a common parasite of Potentilla ; Pyreno- 
peziza medicaginis Fckl. The last of these three species has already 
been shown by the writer (Jones, 1918) to be the ascigerous stage of 

1 In compiling this host list, the fungus found on species of the genus Trifolium is 
regarded as Pseudopeziza trifolii (Biv.-Bern.) Fckl., while the fungus on species of the 
genus Medicago or closely related genera is regarded as P. medicaginis (Lib.) Sacc. 
Owin<r to the fact that the two fungi have frequently been regarded as one, the fungus 
on Medicago and its relatives has often been reported as P. trifolii. 



6 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

Sporonema phacidioides Desmaz. The writer has not been able to 
discover any adequate reason for the inclusion of the other two 
names. 

SYNONOMY OF PSEUDOPEZIZA MEDICAGINIS. 

The first collection and description of Pseudopeziza upon a species 
of the genus Medicago were made by Madam Libert (1832, fasc. 2, 
no. 1T6) under the name of Phacidiurw medicaginis. The host was 
Medicago wUdenowii, now known as Medicago lupulina wUdenowii. 
Later, when Desmazieres (1841) found Pseudopeziza upon alfalfa he 
assumed that it was identical with the species described on Medicago 
wUdenowii. His assumption has not been seriously questioned. 

In 1883 Saccardo (1883, no. 1300, 1301) transferred this species 
to the genus Pseudopeziza which Fuckel (1870) had established 
with Pseudopeziza trifolii as the type species. As soon as the two 
fungi were brought together in the same genus their similarity 
raised the question whether they were not identical. Briosi (1888) 
compared the fungi as the}' occurred on several species of Trifolium 
and Medicago and failing to find sufficient morphological difference 
between them to justify retaining them as distinct species advised 
that Pseudopeziza on alfalfa be called Pseudopeziza trifolii forma 
medicaginis. This usage has been followed by Rehm (1892, p. SOT- 
SOS) and appears to have been generally accepted by mycologists, 
many of whom drop the form name altogether. Plant pathologists, 
on the other hand, have found it more convenient to retain the two 
names, though in most texts it is noted that possibly or even prob- 
ably the two species are identical. The writer believes that the fol- 
lowing pages present adequate evidence that the fungi on the two 
hosts are separate and distinct species. 

COMPARATIVE MORPHOLOGY OF THE FUNGI. 

The apothecia of both these species of Pseudopeziza arise in a deli- 
cate stroma beneath the epidermal layer of the leaf. The apothecia 
on alfalfa are usually solitary, except on overwintered leaves, where 
several clustered apothecia may develop on a stroma. On red-clover 
apothecia are sometimes clustered. The hymenial layer when first 
developed is covered with a thin stromatic stratum of small rounded 
cells, the outer layer of which may develop thick dark-colored walls. 
This stroma usually remains adherent to the epidermis when this is 
ruptured by the developing asci. 

As the hymenial layer develops, the stroma from which it arises 
becomes thicker, forming in and among the collapsing leaf cells. The 
epidermis is ruptured, the hymenium is raised above the surface of 
the leaf, and after the spores have been largely discharged and the 
hymenium has shrunken the recurved flaps of the torn epidermis 
become conspicuous around the apothecium. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 



Under favorable conditions apothecia may reach If mm. in di- 
ameter, but are usually 1 mm. or less. Asci are 60 to TO microns 
long, and about 10 microns in diameter. Paraphyses are slightly 
longer than the asci, nonseptate, and swollen at the ends. 

Ascospores of the two species (figs. 1 and 2) show slight differ- 
ences in size, those of Pseudopeziza trifolii being larger. The 
spores of each species have shown small variations in measurement 
when they were obtained under different conditions affecting their 
discharge. The most important of these variations has occurred 
when spores are obtained from apothecia which are drying rapidly. 
Under these conditions discharge is greatly accelerated, and. the 
number of spores of smaller size is increased. Therefore, in order to 
obtain comparable measurements certain precautions were always 
taken to obtain spores of the same degree of maturity. Fruiting cul- 
tures of the fungus or leaves bearing apothecia were placed in the 
cover of a Petri dish over a layer of clear agar. After about 10 
hours, when the discharge 
of spores was apparently 
proceeding at a uniform 
rate, the cover of the dish 
was turned about so that 
the spores now fell on a new 
portion of the agar surface. 
After half an hour a considerable number of spores were usually 
found on the agar. The cover was then removed from the dish, a 
small drop of water and a cover glass were placed on the area on 
which the spores were scattered, and measurement was made as 
rapidly as possible. 

When a large number of spores have been measured to the nearest 
micron and the spore lengths arranged, as shown in Table II, it has 
always been found that the number of spores of Pseudopeziza medi- 
caginis which measure 10 microns and less constitute more than 
half the total, while in the case of P. trifolii the number of spores 
which are 11 microns and longer constitute more than half the total. 
Table II presents a typical comparison of the measurement of 100 
spores of each species. 




C3> 

Fig. 1. — Ascospores of 
Pseudopeziza trifolii. 
X 600. 



Q 



%> 



Fig. 2. — Ascospores of 
Pseudopeziza medica- 
' ginis. X 600. 



Table II. — Comparison of the lengths of 100 spores each of Pseudopeziza medi- 
caginis and Pseudopeziza trifolii, measured to the nearest micron. 



Spores of — 


Length (microns). 


8 


9 


10 


11 


12 


13 


14 


Pseudopeziza medicaginis number. . 

Pseudopeziza trifolii do 


7 


26 
3 


33 
26 


24 
46 


10 
18 






6 


1 









8 BULLETIN 759, IT. S. DEPARTMENT OF AGRICULTURE. 

By careful comparison in this manner it has been found possible to 
distinguish between the two species on the basis of spore measure- 
ment alone. 

In addition to the difference in size, there is a difference in shape 
that is discoverable by the examination of many spores — a difference 
that does not significantly appear in measurement. Some of the 
spores of Pseudopeziza trifolii are slightly flattened on one side. 
When the flattened side is seen in profile the spore has a somewhat 
pointed appearance. The occurrence of occasional pointed spores 
(fig. 1) is a distinguishing feature of this species. 

Spores from dried specimens have not been found satisfactory for 
comparative measurement. Unless the collection is made just before 
the apothecium is completely mature, nearly all of the spores are un- 
avoidably discharged during drying. The few remaining are likely 
to be found much shrunken. 

MORPHOLOGICAL CHARACTERS IN CULTURE. 

MYCELIUM. 

In culture these fungi preserve the same general characteristics 
that they show on the host plant. The mycelium radiating from the 
germinating spore or group of spores soon produces a stroma at the 
center. Thereafter this stroma is surrounded with a narrow fringe 
of hyphsB, which never advance far beyond the stroma. When the 
fungi are grown from spores on the same nutrient substratum, differ- 
ences in the character of the mycelium can be noted. That from 
Pseudopeziza medicaginis branches earlier than that of P. trifolii' 
most of its branches come off at an acute angle, while those of P. 
trifolii come off somewhat regularly at a right angle, or occasionally 
at an obtuse angle. 

CONIDIUMLIKE STRUCTURES. 

Although no conidia have been found in nature, conidiumlike struc- 
tures occur regularly in culture and are a feature by which cultures 
of the two species can be most easily distinguished. They arise from 
the ends of branches or from the distal ends of somewhat swollen 
cells. They measure 5 to 8 by 3 to 5 microns. They occur most abun- 
dantly when the ascospores are germinated on clear agar to which 
no nutrient has been added, appearing in about three days in the 
case of Pseudopeziza medicaginis and somewhat later on mycelium 
of P. trifolii. On mycelium of the first fungus they are produced in 
great abundance before the end of the first week, though the mycelium 
from different spores or groups of spores produces them in varying 
amount. (Fig. 3.) The mycelium may grow but little, becoming 
thickly covered with the conidia, or it may grow more freely with 
but a few conidia at the ends of short branches. Rarely are they 



LEAF-SPOT OF ALFALFA AND BED CLOVER. 



9 



absent. In striking contrast is the scarcity of these spores on my- 
celium from spores of P. trifolii. (Fig. 4.) Never are they pro- 
duced in great numbers, and frequently they are entirely absent from 
all but a few fungous colonies. Thus, the striking abundance of these 
structures on mycelium of P. medicaginis and their scarcity on 




Fig. 3. — Mycelium and eonidiumlike structures developing from ascospores of Pseudo- 
peziza medicaginis on agar agar. X 400. 

mycelium of P. trifolii furnish^ an easy morphological distinction 
between the two species. 

REPORTED CONIDIAL STAGES OF THESE FUNGI. 

It is a matter of some interest to note that all the studies of 
Pseudopeziza on alfalfa and clover 
which have been made by European 
mycologists and pathologists with 
but a single exception (Briosi, 
1888) have contained a discussion 
of an associated conidial stage. 
Thus at least three, perhaps four, 
imperfect fungi have been assigned 
to this role in addition to the coni- 
diumlike structures which are pro- 
duced in culture. A summary of 
the evidence on the basis of which 
the association of these conidial 
stages has been made is here given. 

The first of these fungi to be 
regarded as a conidial stage of Pseudopeziza medicaginis was Sporo- 
nema phacidioides Desm. Since the writer has a previous article 
(Jones, 1918) traced the development of the purely observational 
evidence on which this association was based and has shown that this 
Sporonema is the conidial stage of Pyrenopeziza medicaginis Fckl., 
no further discussion is necessary here. 
89950°— 19 2 




Fig.- 4. — Mycelium developing from an 
ascospore of Pseudopeziza trifolii on 
agar-agar. X 400. 



10 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

The second suggestion that Pseudopeziza produces conidia comes 
from the work of Brefeld (1891, p. 325). In the course of his study 
of Pseudopeziza on both clover and alfalfa he germinated the asco- 
spores. The cultures thus obtained of these fungi behaved alike. 
Very little mycelium was produced. After about 14 days conidia 
were cut off laterally from certain threads and from the ends of side 
branches. These conidia were not observed to germinate. After 
describing them, Brefeld refers them to the conidia described by 
Tulasne (probably referring to the conidia of Sporonema phacid- 
ioides), but he does not state whether he regards his conidia iden- 
tical with those described by Tulasne or not. The structures which 
Brefeld describes and figures as conidia appear to be identical with 
those already described as occurring in culture. 

The third and most extended reference to a conidial stage of 
Pseudopeziza medicaglnis is that of Voges (1909). In the course of 
a study of an outbreak of the disease in fields under his observation 
in Germany, he reports that he finds closely associated with the fruit- 
ing disks of Pseudopeziza on living leaves the pycnidia of a Phyllo- 
sticta which does not appear to him to belong to a previously de- 
scribed species. Unfortunately, his own description of this Phyllo- 
sticta is so meager that it does not serve to identify it. He states that 
the spores are differentiated into two f orms — a smaller 1-celled spore 
and a larger, often 2-celled spore. No mention is made of any 
attempt to determine whether or not the two types really belong to 
the same fungus or not, nor does he explain why such a fungus should 
not be called Ascochyta rather than Phyllosticta. 

Voges next attempts to identify the Phyllosticta with Pseudope- 
ziza by cultural methods. He places fragments of Pseudopeziza fruit 
disks on a nutrient substratum. When this is done in March and 
October, no results are obtained ; but in June he gets a fungus on his 
plates which first produces aerial conidia and later pycnidia like 
those previously found on the leaves. Inoculations made on alfalfa 
leaves with these leaf-spot cultures produced typical Phyllosticta 
spots. Inoculation of alfalfa leaves with fragments of Pseudopeziza 
fruiting disks gave like results. Consequently he concludes that the 
Phyllosticta and Pseudopeziza are identical and that Pseudopeziza 
has three spore forms — aerial conidia, conidia in pycnidia, and asco- 
spores. Finally he inoculates clover leaves with fragments of his 
Phyllosticta culture and finds that t} 7 pical spots bearing Phyllosticta 
spores are produced. Hence he concludes that the Pseudopeziza on 
alfalfa must be identical with that on clover. 

Even if these results of the few experiments which he performed 
are accepted at their full value, the conclusions which he draws are 
manifestly not justified. In the first place, the fact that he was 
unable to get cultures of his Phyllosticta from Pseudopeziza spots. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 11 

except at a certain period in the summer is not adequately explained 
by his extraordinary theory of a periodicity in the vegetative vigor 
of the fungus. In the second place, inoculations with this Phyllo- 
sticta whether upon alfalfa or upon clover produced lesions which 
bore only the pycnidia of the Phyllosticta, never the apothecia of ther 
Pseudopeziza. These cultures obtained under doubtful conditions 
produced ascospores neither in culture nor as a result of inoculation. 
Thus, the evidence which Voges presents, judged entirely by itself, 
does not prove or even clearly indicate that he ever had Pseudopeziza 
in culture. It does not appear, however, that the work of Voges 
has been widely accepted, at least not in America, even though the 
report of his work as presented in the Experiment Station Record x 
is incorrect or misleading in almost every detail, causing his conclu- 
sions to appear much more justifiable than when they are read in 
the original article. 

In the same year that the article by Voges "was published Voglino 
(1009, pp. 226-228) in Italy presented evidence which he believed 
indicates that Gloeosporium caulivormn Kirch, or G. trifolii Pk., 
which in his opinion may be identical with it, is the conidial stage 
of Pseudopeziza on Trifoliv/rn pratense. His evidence was obtained 
both from observation and from cultures. In a certain field consid- 
erably injured by Gloeosporium he finds apothecia of Pseudopeziza 
trifolii developing in close association with the acervuli of the 
Gloeosporium. Later he makes cultures from conidia obtainetl from 
stems on a clover decoction with gelatin, and in a single culture he 
found after 30 days three apothecia of a fungus which he assumes 
to be a Pseudopeziza. On the basis of this evidence he decides 
that the Gloeosporium must be the conidial stage of Pseudopeziza. 

The account which Voglino gives of his work is very brief and 
bare of details. No mention of inoculations is made. No descrip- 
tion of the Pseudopeziza which he regards as Pseudopeziza trifolii 
is given. It is not clear from his account that he obtained a pure 
culture. Therefore his results can hardly be regarded as having 
more than a suggestive value. 

This review of European literature brings us to the conclusion 
that, with the possible exception of the description by Brefeld of 
conidia in culture, there is no conclusive observational or experi- 
mental evidence that either of these Pseudopezizas has an associated 
condidial stage. 

PHYSIOLOGY OF THE FUNGI. 

ISOLATION OF THE FUNGI. 

Efforts to isolate these fungi by ordinary methods were continued 
for a long time without avail. The first success was obtained by 

1 Experiment Station Record, v. 22, no. 7, p. 648. 



12 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

taking advantage of the fact that when the ascospores are dis- 
charged from the ascus they are thrown several millimeters verti- 
cally. Kepeated trials showed that if fresh leaves were used, spores 
could sometimes be obtained in considerable numbers on an agar 
surface placed over the apothecia without bacterial or fungous con- 
tamination. This work of collecting spores was best carried out in 
a Petri dish in which a layer of very clear agar had been poured. 
The dish was inverted and the leaf bearing the apothecia supported 
2 or 3 millimeters below the agar. After a short period the area over 
the apothecia was marked, the dish turned, and examination made 
with the low power of the microscope to determine whether the 
requisite number of spores were present. The difficulty would have 
been lessened had a few spores been sufficient for the development 
of a culture, but experience soon showed that a large number of 
fungous colonies crowded together developed better than a few. 
When several areas on the plate had been scattered with spores, the 
leaf bearing the apothecia was transferred to another plate. By using 
the utmost care and exposing a large number of plates a few could 
be obtained without contamination or with so small a number of 
foreign organisms that they could be cut out with a sterile needle. 
After a plate had been observed until it appeared certain that no 
foreign organism was present, it was found advisable in order to 
prevent dr}dng to cut out the area bearing the developing fungous 
colonies and transfer them to agar slopes in test tubes. 

In the course of experiments with cultures made in this way the 
first culture of Pseudopeziza from alfalfa to produce apothecia was 
obtained. The spores were discharged on an alfalfa-agar plate on 
October 5, and the agar was transferred to a water-agar slope on 
October 22. The ascospores were being produced on November 6. 
At first it was assumed that the fungus had been starved into fruiting 
by this process, but later work does not indicate that this was the 
case. Fruiting cultures can be obtained most readily by transferring 
the developing fungous colonies as soon as they become macroscopic 
from the water-agar plates to oatmeal-agar slopes. In this way the 
fungus was isolated six times in the autumn of 1914 and once in the 
autumn of 1916. 

Pseudopeziza was isolated from red clover in the same way as 
from alfalfa. Two isolations were made of this fungus in 1915, one 
from clover leaves collected by Prof. H. H. Whetzel at Ithaca, N. Y., 
and one from clover collected in Door County, Wis. 

A later successful reisolation of this fungus from plants inoculated 
in the greenhouse suggests that it may not always be necessary to 
employ this tedious process. In the instance referred to, diseased 
leaf fragments were cut from clover leaves two weeks after inocula- 
tion. • The fragments, each bearing from one to three infections, were 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 13 

dipped in 50 per cent alcohol and placed in a solution of 1 part of 
bichlorid of mercury in 1,000 parts of water for 1 to 1^ minutes. 
After washing, the fragments were placed separately on slopes of 
2 per cent water agar. After these cultures had been kept three 
weeks at 17° to 19° C, small tufts of mycelium emerged from the 
fragments which had remained free from contamination. The frag- 
ments were then transferred to oat agar. Apothecia appeared two 
weeks later, and cultures were started. Success in this instance seems 
to be due to the relative freedom of these greenhouse plants from 
fungi which quickly enter the host tissue that has been killed by the 
parasite. 

CULTURAL CHARACTERS OF SPECIAL MEDIA. 

Only a few of the more common media on which the fungi grow 
most readily and show the most striking differences are selected. 
In connection with these descriptions the following facts regarding 
the method of making cultures and their habit of development should 
be kept in mind : 

(1) New cultures are started by placing near the top of an agar slope a 
fragment of a culture which is producing and discharging ascospores abun- 
dantly. The position of the slope should be changed from time to time to insure 
a somewhat uniform distribution of the spores over its entire length. After 
from one to four days the original transfer may be removed to another slope 
and thus serve to start a number of cultures successively. 

(2) The small fungous colonies which arise fruit better and earlier when 
closely crowded together. Yet excessive crowding may delay fruiting. 

(3) Apothecia appear in three to five weeks at favorable temperatures. 
After a period of active spore production lasting from one to two weeks, fur- 
ther spore production takes place only occasionally. Transfer of the stroma 
to new media increases the likelihood of its occurrence but does not insure 
it. The stroma itself continues to grow very slowly. 

Oatmeal, agar. — As a culture medium oatmeal agar has proved to be 
the most useful for general culture work, because upon it ascospores 
are produced in greatest abundance. The following description 
applies to cultures kept at 20° to 22° C. 

Pseudopeziza medieaginis : The first evidence of growth appears 
about one week after spores are discharged upon the medium. At 
this time the surface of the substrate appears roughened as though 
pushed up into a multitude of minute flat cones. In about two 
weeks the cones are increased in size and show a rusty brown color at 
the center. The color becomes darker at the center of the cone, 
surrounded by a rusty rim. Soon the dark-brown color covers the 
entire slope. If the colonies are not sufficiently close to touch each 
other in three weeks, the color is darker than if the colonies merge. 
At the end of three or four weeks apothecia appear at the center of 
the stromata as small grayish white, often glistening gelatinous 



14 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

masses, very small at first, but often extending until they merge. 
The apothecium is merely a rounded, sometimes flattened, firm mass 
of asci and paraphyses which crumbles when crushed (PI. Ill, B). 
The gray color of the mass becomes darker with the increased age 
of the culture, and finally is dark brown or almost black. The rusty 
brown color that is seen early in the development of the culture is 
usually found to have permeated the agar slightly by the time the 
fruiting stage is reached. A greater amount of color is usually 
found in cultures which have relatively few colonies and which 
fruit but little. Retardation of the growth of the cultures by low 
temperature appears to allow the color to diffuse farther through the 
agar. In cultures which grow rapidly, the yellow color may be 
diffused only in the upper part of the slope where the layer of agar 
is thin. 

Pseudopeziza trifoUi: The development of Pseudopeziza trifolu 
on this culture medium differs from that described for P. medicaginis 
only in the details here stated. The color which develops in the strO- 
mata as they develop is dark gray, becoming black, with no trace of 
brown. The fungous colonies appear a little more vigorous and co- 
alesce into a more solid crust on the surface of the medium than those 
of P. medicaginis. The fruiting structures appear more typical (Pi. 
Ill, ^1), being flat on top, but are not surrounded by a wall. The 
substrate never becomes discolored. 

Potato-dextrose agar. — Cultures of both fungi grow very rapidly 
on potato-dextrose agar. When the colonies are much crowded the 
leathery surface growth becomes crumpled. 

Pseudopeziza medicaginis : Color at the end of four weeks brown, 
sprinkled with a few black stromata; substrate decidedly colored; 
apothecia produced in small numbers. 

Pseudopeziza: Culture coal black, with slight amount of frosty 
mycelium. There is a slight staining of the substrate. 

Lima-bean agar. — Pseudopezlza medicaginis: Growth yellowish in 
varying degree ; no fruiting observed ; substrate shows discoloration. 

Pseudopezlza trifolu: The growth is a rough black mat with a little 
white mycelium. The substrate shows slight discoloration. 

Corn-meal agar. — Pseudopeziza medicaginis: Growth not vigor- 
ous; colonies scattered, black, and distinctly raised from the sur- 
face, which appears as though sprinkled with coarse black pepper; 
apothecia minute and scattered. 

Pseudopeziza trifolii: Growth black, almost submerged in the sub- 
strate; colonies tend to remain separate, though the slope appears a 
solid black color; scattered minute apothecia occur. 

Liquefaction of gelatin. — Both fungi cause rapid liquefaction of 
gelatin. 



Bui. 759, U. S. Dept. of Agriculture. 






Plate III. 



Js^ ^ 




Fruiting Cultures of Pseudopeziza. 

A, Fruiting culture of Pseudopeziza trifolii. X 15. approximately. B, Fruiting culture of Pseudopeziza 
medicaginis from alfalfa. X 15, approximately. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 



15 



SPOKE DISCHARGE. 



The discharge of ascospores has been observed frequently under the 
microscope when apothecia have been crushed in water. The ascus 
slowly becomes longer and of greater diameter, forcing the spores 
forward in a more or less oblique biseriate position. The increase in 
diameter of the ascus of Pseudopeziza medicaginis appears to be 
somewhat greater than in the case of P. trifolii, thus allowing 
the spores to come more nearly into a biseriate position. "When 
the limit of resistance of the ascus wall has been reached, the end rup- 
tures, allowing the spores to be expelled in a column. When dis- 
charge takes place under water the spores show no tendency to re- 
main together, but when they are discharged in air they show a ten- 
dency to remain in pairs. This pairing of the spores is probably due 
to the adhesive quality of the spore wall, a quality which is also 
shown by the tenacity with which the spores adhere to the cuticle of 
a leaf. 

It is interesting to note that the tendency to remain in pairs is not 
shown equally by spores of the two species of Pseudopeziza. It is 
somewhat more marked in the case of Pseudopeziza medicaginis. 
This may be due to the fact already cited that the spores of this 
species are brought more clearly into biseriate position before dis- 
charge takes place. In order to determine this difference, spores were 
collected on water agar as though for measurement. The spores in 
each group in selected miscroscopic fields were counted until, the 
grouping of 1,000 spores had been determined. The results obtained 
at various times are shown in Table III. 



Table III. — Grouping of ascospores of Pseudopeziza medicaginis and Pseudo- 
peziza trifolii when caught on an agar surface after being discharged 
normally from cultures. 



Spores of — 


Spores in group — 


1 


2 


3 


4 


5 


6 


7 


8 


Pseudopeziza medicaginis: 

First lot . number . . 

Second lot do 

Third lot do. . . . 


60 
66 
58 


238 
342 
356 


19 
8 
6 


43 
30 
33 


7 
4 
6 


19 
9 

4 


2 


9 
4 
1 


Total 


184 


936 


33 


106 


17 


32 


2 


14 




Pseudopeziza trifolii: 

First lot , number . . 

Second do 

Third lot do 


133 

272 
242 


215 
189 

185 


39 

48 
54 


28 
22 
19 


16 

7 
10 


11 
4 
9 


2 
5 
2 


6 
3 

4 




Total 


647 


589 


141 


69 


33 


24 


9 


13 





It will thus be seen that while in the case of Pseudopeziza medi- 
caginis each spore group containing an even number of spores is 
greater than the preceding or following group, in the case of P. 



16 



BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 



trifolii there is almost a regular decrease of the number of groups 
from those containing one to those containing eight. Sufficient evi- 
dence has not been collected to determine whether the same clean-cut 
difference in the behavior of the two species appears when the 
spores are discharged from apothecia on living leaves. 

SPORE GERMINATION. 

In all the more obvious features of germination, the ascospores of 
the two fungi behave alike. They germinate readily when they have 
been discharged naturally upon a suitable moist surface; but germi- 
nation is infrequent if the spores have been crushed out of the asci 
or if they are submerged in water. A single germ tube emerges from 
any point in the circumference of the spore, except that germination 
from the end has been observed only in the case of a few spores of 
Pseudopeziza trifolii. Under the best conditions that have been 
found, the proportion of germination is usually from 30 to 50 per 
cent. The vigor of germination varies greatly. Many of the spores 
which push out short germ tubes cease growth promptly, while a few 
develop vigorous germ tubes. 

Table IV. — Time required for the germination of ascospores of Pseudopeziza 
medicaginis and Pseudopeziza trifolii and rate of growth of the germ tube 
for three days at constant temperatures. 

[A plus sign indicates that the germ tube could be seen emerging from the spore, but that it did not reach 
a length equal to half the length of the spore. The figures in the body of the table represent the estimated 
length of the germ tube in terms of the length of the spores.] 





Pseudopeziza medicaginis. 


Pseudopeziza trifolii. 


Temperature (°C). 


4 
hours. 


8 
hours. 


12 
hours. 


24 
hours. 


48 
hours. 


72 
hours. 


4 

hours. 


8 
hours. 


12 
hours. 


24 
hours. 


48 
hours. 


72 
hours. 


2.5 to 3 








0.5 
.75 
.75 

1.5 

1.5 

1.5 

2.5 

2.5 

1 
( 2 ) 


1 

1.5 

1.5 

3 

3 

5 

5 

4 

2 

m 


2 
2 
2 

4 
4 








+ 
0.75 

.5 
1.5 
1.5 
2 

2.5 
1 

+ 

( 2 ) 


1 

1.5 

1.5 

3 

3.5 

5 - 

5 

(') 


2 


6to7 






+ 
+ 
0.5 
1 
1 
1 

.5 
+ 
( 2 ) 






+ 

+ 

0.5 

1 

1 

1 

+ 


2 


9 to 10 




+ 

+ 

0.5 
.5 
.5 

+ 


+ 

+ 
+ 


+ 
+ 
0.5 
.5 

.5 


2 


12 




4 


16 


+ 
+ 
+ 


5 


21 to 22 




24 to 25 




27 to 28 


(') 


29 




( 2 ) 








30 


( 2 ) 


( 2 ) 


( 2 ) 


( 2 ) 


( 2 ) 


( s ) 


( 2 ) 







Spores disintegrate. 



2 No germination. 



The one significant difference that they show is found in the fact 
that the spores of Pseudopeziza medicaginis will continue to germi- 
nate at a slightly higher temperature than those of P. trifolii. In 
order to test this relation of temperature to germination, spores were 
discharged naturally upon an agar surface with all the precautions 
necessary to secure mature spores previously described in obtaining 
spores for measurement. The agar used in all cases consisted of 2 
per cent of agar-agar in water carefully cleared and filtered. In 
order to insure identical conditions for the two species in each test, 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 



17 



transfers of both were made to the cover of the Petri dish contain- 
ing the agar. The spores were discharged simultaneously for 1 to 1\ 
hours. If at the end of this time sufficient spores were present, the 
cover containing the cultures was transferred to another dish and the 
spores placed at the desired temperature. Petri dishes to the number 
of 72, nearly all of them containing spores of the two fungi, were 
incubated in this way. The results are given in Table IV. 

This table shows that the two species germinate with almost ex- 
actly the same degree of vigor at each temperature, except that the 
spores of Pseudopeziza trifolii cease to germinate, or, perhaps more 
exactly, to make growth after the initial stages of germination, at a 
slightly lower temperature than those of P. medwagmis. Although 
the difference here may seem slight — almost within the limit of ex- 
perimental error — it has been found constant and definite in repeated 
tests. The possible significance of this fact in connection with the 
difference in geographic range of the two fungi will be considered 



later. 



EFFECTS OF TEMPERATURE ON SPORE PRODUCTION. 



When cultures of the two fungi are placed at a series of constant 
temperatures they appear to show constant differences in the time 
required for spore production and in the abundance of spore pro- 
duction at each temperature. At the outset it must be stated that 
limitations inherent in the method of starting cultures cause wide 
variation in the behavior of the cultures of each species. This diffi- 
culty is partly overcome by using several cultures and by repeating 
the work. Even then the results must be regarded as being sug- 
gestive rather than accurate. The time required for the production 
of ascopores as obtained in two trials is given in Table V. Three 
cultures of each fungus were used, and the earliest time at which 
the discharge of ascospores could be demonstrated in any of the three 
cultures was recorded. The three highest and the lowest temperature 
rarely varied more than one degree, and the remaining temperatures 
varied only half a degree. 

Table V. — Time required for the production of ascospores of Pseudopeziza 
medicaginis and Pseudopeziza trifolii at constant temperatures. 



Ascospores of— 


Temperatures (°C). 


8 


9 


11 


12.5 


13 


14 


15 


16 


18.5 


20 


21.5 


24 


Pseudopeziza trifolii days . . 

Pseudopeziza medicaginis do 


82 
82 


53 


52 


30 
58 


43 


30 

52 


36 
49 


30 
36 


20 
16 


16 
14 


16 
14 


21 









Table V shows that below 14° C. the behavior of Pseudopeziza 
medicaginis was erratic, rarely fruiting at all. Only one culture 
89950°— 19 3 



18 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

fruited at 8° C. in one of the trials. At and below 16° P. trifolii 
requires a shorter time for the production of spores than P. me'di- 
caginis, while above this temperature the condition appears to be 
reversed. But the most striking differences in the behavior of the 
two fungi are not shown in the table. These differences lie in the 
length of the spore-producing period and in the abundance of the 
spores produced. Although P. trifolii is under most circumstances 
the more prolific spore producer, this preponderance is greatly in- 
creased at and below 18° C. Here abundant fruiting may occur for 
two weeks and longer, while cultures of P. mcdicaginis fruit very 
meagerly and only for a short period at these temperatures. Above 
16° P. medicaginis begins to fruit more abundantly, reaching its max- 
imum at temperatures a little above 20° C. Thus, the optimum tem- 
peratures for the fruiting of these fungi may be judged roughly as 
about 13° to 22° C. for P. trifolii and 16° to 24° for P. medicaginis. 

RESISTANCE OF THE SPORES TO DESICCATION. 

In order to determine whether ascospores discharged from the 
ascus might be able to live over winter on seed or debris carried with 
the seed, it was desirable to test the resistance of discharged asco- 
spores to periods of drying of such duration as they would be obliged 
to endure on the seed. Owing to the slow growth of the fungus after 
germination and the limitations of conditions under which spores 
will germinate at all, it is obvious that the spores must be dried and 
germinated under such conditions that all other organisms will be 
excluded ; that is to say, the entire process must be carried out under 
conditions of pure culture. 

Obviously, the preferable method would be to dry the spores on 
the seed itself. In order to do this, sterile seeds were necessary. The 
difficulty of obtaining such seeds which were certainly free from any 
residual effect of the sterilizing agent on the seed coat was so great 
that it was finally abandoned. 

Preliminary tests with plaster of Paris blocks as the conveyor for 
the spores during desiccation were so satisfactory that they were used 
exclusively. Thin blocks small enough to slip into a test tube easily, 
were sterilized by heat and placed beneath cultures which were dis- 
charging spores actively. After a period of 8 to 12 hours the blocks 
were placed in sterilized test tubes. These were stored, some in a 
glass case in the laboratory and some outside a north window. 

From time to time one or more of these blocks were placed on 
water agar to which a small amount of alfalfa-leaf decoction had 
been added. The amount of the decoction appeared to make no ma- 
terial difference up to any amount that could be added to 3 per cent 
agar without causing it to lose its ability to solidify upon cooling. 
Other culture media were tried, but none gave as prompt a result 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 



19 



as this. The red-brown color of the Pseudopeziza stroma produced 
by this medium on these white blocks (PI. I, B) could be recognized 
at an earlier date than the pale-colored growth which developed 
when other media were tried. 

Spores of Pseudopeziza trifolii were tested in the same way with 
those of P. medicaginis, though not as extensively. The results are 
given in Table VI. 

Table VI. — Viability test of ascospores of Pseudopeziza medicaginis and Pseu- 
dopeziza trifolii viable when dried on plaster of Paris blocks in the laboratory 
and out of doors. 



Place and date of ending 
desiccation. 


Time 
(days). 


Results. 


Place and date of ending 
desiccation. 


Time 
(days). 


Results. 


PSEUDOPEZIZA MEDICAGINIS. 

In laboratory: 

Feb 1, 1915 


32 
36 
37 
38 
39 
48 
49 
63 
66 
72 
76 
76 
79 
85 
93 
94 
100 
104 
210 
357 

27 
31 

52 
77 
78 
106 


+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 





+ 




+ 
+ 



+ 
+ 

4- 


PSEUDOPEZIZA MEDICAGINIS— 

continued. 

Out of doors — Continued. 

May 20, 1916 


120 
108 
159 
212 
303 
329 

36 
63 
64 
112 
113 
120 
258 
256 

28 
25 
104 
107 
265 
265 




Jan 23, 1915 




Dec. 3, 1915 


Feb. 15, 1916 




Feb 1, 1915 


Apr. 7, 1916 




Dec 9, 1916 


May 20, 1915 . . 





Dec 16, 1915 


Sept. 1, 1916... 




Feb. 20, 1915 


Do 





Mar 26, 1915 


PSEUDOPEZIZA TKIFOLII. 

Jn laboratory: 
Jan 17, 1916. 




Jan 5, 1916 




Jan 12, 1916 




Jan 17, 1916 






+ 


Jan. 22, 1915 


Mar. 7, 1916 


Jan 1, 1915 


Feb 15, 1915... . 




Aug. 26, 1915 


Apr. 6, 1916 





Do 


May 15, 1916 




Apr. 11, 1915 


Apr. 20, 1916 





Feb. 12, 1916 


Sept. 1, 1916 





Sept. 1, 1916 


Do 





Jan. 15, 1916 


Out of doors: 

Jan. 17, 1916 




Out of doors: 


+ 


Dec. 9, 1915 


Do 


+ 


Dec. 16, 1915 


May 4, 1916 


+ 


Dec 22, 1915 


Apr. 7, 1916 





Jan 17, 1916 


Sept. 2, 1916 





Jan. 13, 1916 


Sept. 1, 1916 





Feb 15, 1916 















From these results it appears that drying alone can not be de- 
pended upon to kill all the spores of either Pseudopeziza in less than 
one year. Severe freezing during drying had no apparent effect. 
If conditions for survival are as favorable on the seed as on plaster of 
Paris blocks, the spores should be able to live from one season to the 
next on the seed. But unless conditions on the seed are more favor- 
able than on the block, they should not be able to survive and 
germinate during a second year. 

PATHOGENICITY OF THE FUNGI. 



METHOD OF MAKING INOCULATIONS. 



In all inoculations that have been made, ascospores alone have been 
used as the inoculum. The conidiumlike structures which have 
been described are produced almost wholly in the substrate, and 
since only rarely will one of them separate from the mycelium, no 



20 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

practicable method for using them has been developed. Since no 
evidence has been found indicating that they occur in nature or that 
they germinate, no great importance attaches to them. 

In the first inoculations an attempt was made to obtain the asco- 
spores in water suspension, but when the apothecia were crushed in 
water usually only a few spores became separated from the ascus. 
The few attempts to make inoculations with such meager spore sus- 
pensions failed. The method finally adopted and used with minor 
modifications in all inoculations reported takes advantage of the 
natural discharge of spores from cultures. If a Pseudopeziza cul- 
ture is removed from the test tube carefully it may be cut into frag- 
ments and placed on a support, where it will continue to discharge 
spores for several days provided it is not exposed to direct sunlight 
or high temperatures. A culture may thus be removed from a test 
tube and placed over a plant upon which the spores will fall. If the 
whole plant is to be inoculated, the culture may be placed in the top 
of a bell jar which is set over the plant and turned from time to 
time to insure a uniform distribution of the spores. 

If single leaves are to be inoculated, the culture or fragments of the 
culture may be placed for a short time over these leaves in succession. 
A more uniform discharge of spores for long periods is obtained in a 
dark room at 16° to 20° C. If inoculations are made in the field, they 
should be made at night or on a cloudy day. The plants may be wet 
with a fine spray before the spores are discharged, or if time permits 
this may be deferred until the spores are on the leaf. In the latter 
case, a larger number of infections are usually secured, due appar- 
ently to the fact that spores falling on large drops of water are held 
from sinking by surface tension and germinate too far from the leaf 
surface to effect penetration. After the inoculated plants are sprayed, 
they should be kept in a moist chamber for at least 12 hours. 

This method has been employed in all inoculations made, unless 
otherwise stated. 

CONDITIONS UNDER WHICH INOCULATIONS WERE MADE. 

If inoculations are to be entirely conclusive in result, the control 
plants must remain free from the disease. It has been found impos- 
sible to keep plants free from leaf-spot for infection experiments 
during the summer at Madison, Wis., where the work was done. This 
has been due to the fact that alfalfa fields are located so close to the 
greenhouse that spores are easily blown in through the ventilators. 
But it has been found that if all diseased alfalfa foliage was removed 
from the greenhouses in the autumn after the ground outside froze, it 
was possible to keep alfalfa plants free from infection with leaf -spot 
during the winter and spring. Therefore all inoculations have been 
made or at least repeated during the winter months. This precaution 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 21 

has not been necessary in the case of inoculations with Pseudopeziza 
trifolii, since the Pseudopeziza leaf-spot has not occurred on any spe- 
cies of Trifolium about Madison during the time this work was in 
progress. The following host plants have been inoculated with pure 
cultures of both species of Pseudopeziza. 

HOST PLANTS INOCULATED. 

Medicago saliva with Pseudopeziza medicaginis. — In seven or eight days, 
under greenhouse conditions, infections begin to show as minute brown spots 
scattered over the foliage. If infection is very abundant these leaves quickly 
die. If only four or five infections are scattered over each leaflet, apothecia 
begin to appear in about two weeks. For instance, one inoculation made on 
December 2, 1915, produced abundant apothecia on the spots by December 15. 
On December 20 spores discharged from apothecia on one of the leaves of this 
plant were cultured, and the fungus was recovered. An inoculation made on 
April 17, 1915, on plants in the field before the natural infection developed 
showed abundant spotting 11 days later. The weather turned cold after this 
date and no fruiting was observed. 

Leaves of all ages are attacked. Leaves which have grown to full size 
appear to develop more abundant infections than leaves which are not full 
grown, but leaves which are yellow and weak do not seem to become infected 
as easily as those which are more vigorous. 

None of the infection experiments performed during three winters has failed 
to develop a greater or less amount of typical leaf-spot. 

Medicago sativa with Pseudopeziza trifolii. — Inoculation experiments have 
been repeatedly conducted parallel with those already cited on alfalfa, using 
plants of different ages. No infections visible to the naked eye have been 
produced. 

Medicago lupulina with Pseudopeziza medicaginis. — Plants of this host have 
never been very thrifty under greenhouse conditions, and therefore not a large 
number have beeen available for inoculation. In no case has any infection been 
obtained. Attempts to secure infection by setting Medicago lupulina plants 
in the garden among alfalfa plants which were heavily infected with Pseu- 
dopeziza medicaginis also failed to produce infection. 

Medicago lupulina with Pseudopeziza trifolii. — Only two inoculations have 
been tried. No infections resulted. 

Melilotus alba with Pseudopeziza medicaginis. — On March 28, 1915, several 
leaves of a vigorous sweet-clover plant were placed beneath fragments of a 
culture of Pseudopeziza medicaginis which was discharging spores. At the end 
of 24 hours the culture was placed over the entire plant, which was kept in a 
moist chamber 48 hours longer. After four days the leaves first inoculated 
showed minute brownish spots. These did not increase in size. After two 
weeks the portions of the leaves bearing the minute spots were embedded in 
paraffin and sectioned. In these sections the brown spots were found to 
consist of dead shrunken cells in which traces of mycelium could be found. 
But this mycelium appeared to be shrunken and dead and not advancing into 
the living cells of the host. From this, it appears possible that under favorable 
conditions P. medicaginis may be able to cause a very slight spotting of sweet- 
clover leaves. 

Trifolium pratense with Pseudopeziza medicaginis. — Inoculations of red- 
clover plants in the greenhouse with pure cultures failed to produce any infec- 
tion. Red-clover plants grown in the garden in close proximity to badly dis- 



22 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

eased alfalfa plants have never shown a trace of this leaf-spot during the two 
years that these diseases have been under observation. 

Trifolium pratense with Pseudopeziza trifolii. — Infection from inoculation 
with spores from pure cultures has been at all times easy under greenhouse 
conditions. Frequently such abundant infection has been obtained that the 
leaves are killed before the spots develop to a fruiting stage. The incuba- 
tion period of the disease appears to be slightly longer than that of Pseudope- 
ziza medicaginis, varying somewhat with the abundance of the infection. If a 
leaf is heavily infected, the individual infections appear to develop more 
rapidly and produce small characteristic killed areas earlier than if infections 
are few. Numerous infections appear in 11 or 12 days, and in the greenhouse the 
leaf frequently dies a few days later. Fruiting bodies have not been observed 
on such leaves, but a reisolation has been made from infections two weeks after 
inoculation. 

Less numerous infections may not show for two weeks or even longer, but 
at the end of three weeks or more they may produce typical apothecia if the 
air of the greenhouse is sufficiently moist. These apothecia have been found 
on leaves nearly or quite dead. In this respect the fungus behaves differently 
than does Pseudopeziza medicaginis. Apothecia on dead leaves are very diffi- 
cult to discover unless the leaf has been in a moist atmosphere for some time, 
when they appear as dark-amber gelatinous masses on the leaf surface. The 
exposure of these gelatinous masses of asci to dry air for even a few minutes 
causes them to discharge the larger part of the spores present and to shrink 
to a minute mass only a little darker in color than the leaf and therefore 
difficult to identify. The best development of apothecia has been obtained by 
placing infected plants outside the greenhouse during protracted periods of rainy 
weather. 

Trifolium liybridum with Pseudopeziza medicaginis. — No success in obtaining 
visible infections has been attained. 

Trifolium liybridum icith Pseudopeziza trifolii. — Of the several plants inocu- 
lated only one survived in a vigorous condition for a sufficient period to show 
infection. This showed an abundant spotting, which was in every way charac- 
teristic of the Pseudopeziza spot on red clover except that the spot appeared to 
be somewhat limited in development by the veins, thus showing a slight ten- 
dency to become angular. No fruiting bodies were produced. Apparently 
infection takes place only under the most favorable circumstances. This plant 
appears to be a much less congenial host than red clover. 

GEKMINATION OF THE SPORES ON THE LEAF. 

While study was being made of the leaf-spot fungus in the host 
tissue it was found that the mode of penetration could be observed 
very readily by decolorizing the leaf soon after inoculation was 
made. This method of study was used, not only to determine the nor- 
mal penetration of these fungi into their own hosts, but to determine 
the relation of these parasites to other closely related plants reputed 
to be hosts of these fungi but upon which infection had not been 
obtained. In case preliminary inoculations failed to give visible 
results it was more simple and rapid to determine whether the spores 
of that fungus could penetrate the host in question and develop after 
penetration had taken place than to conduct other extensive inocula- 
tions. Thus, a study of penetration has formed a part of all inocu- 



LEAF-SPOT OF ALFALFA AND EED CLOVER. 23 

lation trials. The results of the two methods of attacking host rela- 
tionships should be considered together. 

METHOD OF STUDY. 

The most of the data given here have been obtained by the follow- 
ing simple procedure : Leaves which have just reached full develop- 
ment are selected for inoculation. A culture of Pseudopeziza known 
to be discharging spores abundantly is supported over the leaf or one 
of the leaflets so that the spores as they are discharged will all fall 
upon it. The leaf may be removed from the plant for studies which 
do not involve a period of more than two days, since results obtained 
from such leaves have always been found by comparison to agree 
with results obtained from leaves attached to the plant. The leaf 
may be sprayed with very fine spray before the spores are discharged 
upon it, but more abundant penetrations are usually obtained if the 
spores are allowed to stick to the leaf before it is wet. When the 
leaf has been kept moistened for at least 12 hours, usually longer, 
it is removed, dropped into a mixture of equal parts of acetic acid 
and alcohol and promptly heated to the boiling point. Leaves which 
are killed promptly in this fashion decolorize in better condition than 
when slower killing takes place in cold acetic alcohol. The acetic 
alcohol is changed until all color has been removed from the leaf. 

The leaf may then be mounted in this liquid on a slide under a 
cover glass and examined under the microscope. The epidermal cells 
should be perfectly clear, and the entire structure of the leaf to its 
very center should be visible. The spores remain attached to the leaf 
during the treatment, and the method of entry and the mycelium 
within the leaf can be clearly seen. No method of staining has been 
found to improve the visibility of the fungus. 

Although this method works best in the case of alfalfa leaves, it 
works well enough with the various clovers to give entirely satis- 
factory results. 

METHOD OF PENETRATION. 

In all of the hundreds of penetrations observed the method of 
entry has invariably been as here stated. The spore is found stuck 
fast to the leaf. The germ tube emerges from the spore either within 
or at the margin of the area of contact of the spore with the leaf and 
passes directly through the cuticle into the epidermal cell. Occa- 
sionally a spore sends out its germ tube along the surface of the 
leaf, but such a germ tube has never been observed to enter the leaf. 
Apparently the germ tube must enter the leaf at the moment of 
emergence from the spore, if at all. Ordinarily there is no per- 
ceptible thickening or alteration of the wall in consequence of this 
penetration. The actual opening appears to be extremely minute. 



24 



BULLETIN" 759, TJ. S. DEPARTMENT OF AGRICULTURE. 



After passing through the wall, the germ tube quickly expands to 
normal size. When it reaches the center of the cell it usually divides 
(fig. 5) into two or three branches, which pass into the adjoining 
epidermal cells or down into the palisade layer. Cell walls do not 
appear to offer any obstruction to advancing hyphse. No marked 
disorganization of the cell contents appears to result from this in- 
vasion until hyphae become very numerous. 

The actual time required for a spore to germinate and transfer its 
contents to the germ tube inside the leaf has not been determined 
accurately, but in most cases it must be less than 12 hours at 18° to 
22° 0. 

This description applies to the method of penetration of Pseudo- 
peziza medicaginis and P. trifolii in their respective hosts. The 
following notes have been made of the penetration of germinating 

spores of these 
fungi in other 
reported hosts 
that have been 
available : 

Trifolium pra- 
tense by Pseudo- 
peziza medica- 
ginis. — Inoculated 
leaves were de- 
colorized in two, 
three and five 
days after inocu- 
1 a t i o n . After 
three days the 
host cells beneath 

many of the germinated spores had yellow granular contents. The yellow 
color made the exact relation of the germ tube to the cell impossible to deter- 
mine. In five days it could be observed that in the case of at least a part of 
these yellowed cells the germ tube had passed through the epidermal cell wall, 
but had not advanced far into the cell. 

Medicago sativa by Pseudopeziza trifolii. — A leaf inoculated on the plant on 
March 2S, 1915, appeared to show penetration in 60 hours. Five leaves inocu- 
lated on December 4, 1916, and decolorized four days later showed many yel- 
lowed cells beneath germinated spores. Suitable fragments were embedded 
and penetrations found in sections. The germ tube had not advanced beyond 
the first cell which it entered. 

Medicago lupulina by Pseudopeziza medicaginis. — Four series of leaves re- 
moved from the plant were tried. Penetrations were noted in 23 hours at 22° C. 
Penetrated cells always show yellow granular contents. Even after 75 hours 
it was doubtful whether the germ tube had advanced beyond the first cell 
penetrated. 

Medicago lupulina by Pseudopeziza trifolii. — One series of leaves was removed 
from the plant. Penetrations were abundant and the penetrated cells yellowed, 




Fig. 5. — Penetration of the epidermis of an alfalfa leaf by the 
germinating ascospores of Pseudopeziza medicaginis. X 800. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 25 

but even after 52 hours the germ tubes had not advanced beyond the cell first 
penetrated. 

Melilotus alba by Pseudopeziza trifolii. — Three days from inoculation a few 
germ tubes were found distinctly inside epidermal cells, but they had advanced 
but slightly. Penetrated cells were not yellowed. 

Melilotus alba by Pseudopeziza mcdicaginis. — The cultures used for inocula- 
tion in this series did not produce many spores. Nevertheless, after four days 
two penerations were found. The penetrated cells were very slightly yellowed. 

Trifolium hybridum by Pseudopeziza trifolii. — No examination was made 
until two days after inoculation. At this time penetrations were abundant 
and easily seen. In a few cases germ tubes had advanced into cells adjoining 
those first penetrated. No yellowing of penetrated cells was observed. 

Trifolium hybridum by Pseudopcziza mcdicaginis. — In 24 hours penetrations 
were abundant, but the germ tubes had not advanced far into penetrated cells. 
Penetrated cells showed no yellowing. 

It appears that the results which are shown above are exactly 
parallel to the results obtained from inoculations. In every case 
where infection in varying degree has been attained, penetration has 
occurred abundantly and the growth of the hyphse within the host 
cells has been rapid without causing discoloration of the cell con- 
tents. In cases where visible infection has not been obtained, the 
relative number of penetrations is usually reduced, but in any case 
growth of the germ tube ceases promptly upon entering the epidermal 
cell. Thus, the resistance which the plants that can not be infected 
offer appears to be due not to any mechanical obstruction to entry, 
but to something within the epidermal cell which prohibits growth. 

These infection experiments supported by penetration studies have 
failed to produce a completely successful infection of any of the 
hosts tried except those from which the fungi were isolated. This 
result is very different from that which - was anticipated from a 
consideration of the host lists. Have we here a group of closely 
similar fimgi highly specialized in their host relationships? 

It is noteworthy that in both host lists only two or three of the 
species of Trifolium are native to America. All the rest have been 
introduced if they occur here at all. Moreover, some of these intro- 
duced hosts which are widely distributed do not appear to be 
attacked by this parasite except in certain limited areas. For ex- 
ample, Pseudopeziza medicaginis has been found on Medicago lupu- 
lina only in New York; P. trifolii has been reported on Trifolium 
repens only by McClatchie (1895) on the Pacific coast 1 and not at 
all on T. hybridum. It is not likely that these fungi have been over- 
looked on these hosts in other localities, and therefore the conclusion 
that they do not always pass to these hosts can hardly be escaped. 
Thus, there is reason to suspect that these two species, as now re- 

1 An excellent collection of Pseudopeziza on Trifolium repens now in the possession of 
the writer was made by Mr. C. W. Hungerford at Olga, Wash., on Sept. 3, 1916. 



26 



BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 



garcled, are made up either of several closely similar species or of a 
group of specialized races. 

Especially in the case of the Pseudopeziza on Medicago lupulina 
the fragmentary evidence indicates a species distinct from that on 
alfalfa. In addition to the evidence already given, the measurements 
of 113 ascospores collected under standard conditions is adduced. 
(Table VII.) 

Table VII. — Lengths of 113 ascospores of Pseudopeziza medicaginis on Medi- 
cago lupulina, measured to the nearest micron. 



Ascospores of — 


Length (microns). 


9 


10 


11 


12 


13 


Pseudopeziza medicaginis number. . 


1 


14 


59 


31 


8 



When Table VII is compared with Table II, it will be noted that 
these spores are even larger than those of Pseudopeziza trifolii. Un- 
fortunately, the cultures which were made of this fungus were de- 
stroyed, and complete evidence of the relationship of these fungi was 
not obtained. 1 Of the other hosts of P. medicaginis, less can be said. 
Field observation indicates that the fungus in Medicago falcata and 
M. hispida denticulata occurs by infection from Pseudopeziza in al- 
falfa. A collection of the fungus has been found in only one in- 
stance each on Melilotus alba and Vicia villosa. An examination of 
a portion of the collection on Melilotus alba reveals a lesion that is in 
all respects similar to that which the fungus causes on other hosts, 
but no asci and spores by which the fungus could be identified were 
found. Certainly the occurrence of the fungus on these hosts is not 
common or of economic importance. No material of Pseudopeziza 
on Onobrychis sativa or any species of Trigonella has been available 
for study. 

Of the host list of Pseudopeziza trifolii only a few species have 
been available for study. The fungus from Trifolium pratense has 
not infected any other species to produce fruiting of the fungus on 
that host. Furthermore, it has not been found fruiting on any other 
host in America except in the one instance already mentioned. Yet 
reports well supported by herbarium specimens indicate that Tri- 
folium repens and T. hybridum are abundantly infected in Italy. 
Whether this infection is by the same species of the fungus or not 
can not be determined at present. 

1 It is of interest to note that Pseudopeziza medicaginis was first described on what is 
now known as the variety ivildenoivii of Medicago lupulina. If it should be shown that 
Pseudopeziza on Medicago lupulina icildenoKii is a distinct species from that on Medicago 
sativa the name Pseudopeziza medicaginis will be restricted to the fungus on the original 
host, and a new species name will be required for the fungus on alfalfa. 



LEAF-SPOT OF ALFALFA A^D RED CLOVER. 27 

LIFE HISTORY OF THE CAUSAL ORGANISM IN RELATION TO 
THE HOST PLANTS. 

AMERICAN STUDIES BEARING ON LIFE HISTORY. 

The great economic importance of the alfalfa crop in America 
has inspired the small amount of work which has been done upon 
this disease with a point of view quite different from that shown in 
the European studies already reviewed. Life history has been studied 
with a view to the possible control of the disease. 

The first work was done by Chester (1891) at the Delaware 
Agricultural Experiment Station. In 1889 twenty plats of alfalfa 
were seeded in different parts of Delaware, with seed from the 
same source. The disease appeared on all of these plats at about the 
sixth week after planting. The plat under closest observation at 
Newark showed a yellowing of the leaves accompanied by black spots 
before Psendopeziza was found fruiting on some of the dark spots. 
Evidently some other disease was associated with Pseudopeziza. 
Chester concludes from this experiment that the disease is carried 
by the seed and next tries a method of disinfecting the seed in order 
to prevent such conveyance. Seed was treated with copper sulphate 
and planted in heat-sterilized soil in cans. The diseases appeared 
on all the plants from these cans. Therefore, Chester concludes that 
the source of the disease must be a general atmospheric infection. 
Unfortunately, in none of his experiments does he give any details 
regarding the proximity of his plats or plants in cans of sterile soil 
to alfalfa which was infected with the leaf-spot and which might 
have been a source of wind-borne spores. 

The only other attempt to study the disease which has been re- 
ported was made by Coombs (1897a) in Iowa. On August 20 alfalfa 
seedlings 3 weeks old grown under bell jars were treated as follows: 
One was left as control, one sprayed with germinating Pseudopeziza 
spores, and one sprinkled with powdered diseased leaves showing 
disease. Next, plants growing in the field were cut back, and after 
the debris was removed, the roots were protected by large bell jars. 
In the next six weeks the plants outside the bell jars became diseased, 
while those inside were healthy. However, when the jars were re- 
moved the plants immediately became diseased. As a result of this 
work Coombs concludes that two things are established: (1) That 
plants are infected by spores from the air and (2) that the disease is 
strictly local and not systemic. 

It will be seen at once that these conclusions are based on a very 
small amount of experimental evidence. Such important factors as 
the high temperature and absence of dew or rainfall inside the bell 
jars do not appear to have been considered as possible conditions 



28 BULLETIN 159, U. S. DEPARTMENT OF AGRICULTURE. 

which might have prevented infection even had the spores heen 
present. 

At this point, it is convenient to mention an English discussion of 
this disease by Iv} T Massee (1914) in which an effort is made to 
throw light on the method by which the fungus is conveyed from one 
locality to another. In this article Miss Massee states that so far as 
England is concerned the dissemination of the disease is due to 
diseased seed which is badly cleaned. She says, " I have recently 
examined a sample of commercial seed and found the fungus present 
in abundance on minute fragments of leaves and calyces, and rarely 
on the seed itself." Unfortunately, Miss Massee does not state the 
methods by which she was able to make the identification of the 
fungus with such certainty on this single sample of commercial seed. 
Even if she was correct in this observation, it still remains to be 
proved that the fungus which she found was alive and capable of 
infecting the plants grown from this seed. 

The many scattered observations of the disease merely contribute 
data regarding distribution, seasonal occurrence, and environmental 
factors. Most of these reports indicate that the disease is worse on 
plants during the first summer of their growth than later, but 
Coombs (1897) speaks of attacks as being worse after the first year. 
Most reports indicate that the disease usually gains headway slowty 
in the spring and becomes worse later in the season, but this is not 
always the case. Stewart, French, and Wilson (1908, p. 384-387) 
speak of the disease as being worse in dry years while most reports, 
especially from drier regions, indicate that the disease is worse in 
wet seasons. 

The facts bearing upon the overwintering of the fungus in the 
field are surprisingly meager. The only definite bit of observational 
evidence is that of Chester (1891), who states that he found live 
asci on leaves in midwinter. Voges (1909) suggests in addition that 
the fungus survives the winter in living leaves. 

After surveying these scattered references in American literature 
we find that there is a general belief that Pseudopeziza medicaginis 
is distributed with the seed and survives the winter on dead leaves. 
This opinion has been reached, not so much as the result of careful 
experimental evidence, which in fact is meager, but more as the 
cumulative effect of the expressed opinions of competent observers 
who have watched its development during a series of years. 

METHOD OF OVERWINTERING. 

The first evidence of the method of overwintering of the leaf-spot 
fungus in the field was obtained in the spring of 1916. On April 11 
overwintered alfalfa leaves showing abundant Pseudopeziza spotting 
were brought into the laboratory and placed in a moist chamber. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 29 

Two days later, when one of the apothecia was crushed out in water, 
asci containing spores apparently mature were found. The leaf was 
then supported over an agar surface. In two hours a large number 
of spores were discharged, which germinated promptly. At this 
time the young alfalfa shoots had hardly emerged from the mulch 
of the debris of the previous season's growth. 

On May 6 spots began to appear on the alfalfa foliage of some of 
the plats under observation. When the spotted leaves were decol- 
orized, characteristic Pseuclopeziza spores with germ tubes penetrat- 
ing the epidermal cells were found in nearly all the spots. 

A search of the overwintered foliage discovered a large number of 
fresh-appearing apothecia developed on leaf areas that had been 
diseased the previous year. When the overwintered leaves were 
placed over agar plates, a large number of viable ascospores were 
caught. 

Fragments of overwintered leaves bearing apothecia were placed 
over ten marked leaves of a healthy alfalfa plant in the greenhouse, 
and the plant was kept in a moist chamber for 24 hours. On May 17 
seven of the ten marked leaves showed more or less of the charac- 
teristic Pseudopeziza spotting. 

Five or six of these overwintered leaves bearing apothecia were 
placed on the ground under a rank growth of alfalfa plants in the 
greenhouse. The plants were then sprayed, but not covered to pre- 
vent evaporation of water from the foliage. On May 21 the alfalfa 
foliage in the vicinity of these overwintered leaves was found in- 
fected with leaf-spot. 

On March 31, 1917, overwintered alfalfa leaves bearing leaf-spot 
lesions were collected in an alfalfa plat. At this time no spores 
could be found in the apothecia. These leaves were kept in a moist 
chamber for a week, care being taken to soak them in water twice 
a day to remove the products of decomposition. At the end of the 
week asci with mature spores had developed in the old apothecia. 

Thus, it appears evident that Pseudopeziza medicaginis survives 
the winter on diseased foliage which escapes decay. When the 
weather becomes sufficiently warm in the spring and moisture is 
provided by protracted rains or the shelter offered by the young 
growing foliage, new asci develop apparently in the old apothecia 
and, in addition, new apothecia are produced around the old one 
(PL II, A). The spores thus produced furnish the primary infec- 
tion in the spring. 

Apothecia producing spores indistinguishable from those of Pseu- 
dopeziza trifolii were found on overwintered clover leaves in the 
spring of 1916 in northern Wisconsin, but since no inoculations were 
made with these spores, tlieir identity was not determined. However, 



30 BULLETIN" 759, U. S. DEPARTMENT OF AGRICULTURE. 

it does not seem unlikely that this fungus winters in the same way 
that has been demonstrated for its close relative on alfalfa. 1 

METHOD OF DISTRIBUTION. 

SUGGESTIONS FOUND IN THE LITERATURE. 

Since the method of overwintering of these fungi has been traced, 
it is now possible to see their entire annual cycle in a field in which 
they have once been introduced. But thus far no information has 
been gained which serves to indicate how they are conveyed into 
new localities in which the host plants are grown for the first time. 
This phase of the problem is of special importance in connection 
with the alfalfa leaf-spot. A large amount of recorded experience 
indicates that this disease appears wherever alfalfa is grown, re- 
gardless of environment. A knowledge of the source of infection in 
these new localities might suggest feasible control measures. 

Scattered through the various discussions of the alfalfa leaf-spot 
are found four suggestions that have been put forth to explain the 
constant appearance of Pseudopeziza on alfalfa in newly seeded 
fields: It is suggested (1) that the fungus is carried with the seed, 
(2) that it is conveyed in soil that is used to inoculate the new field 
with the bacteria producing nodules on the roots, (3) that the fungus 
spores are generally distributed in the air, and (4) that other host 
plants near by furnish the source of infection. Since none of these 
suggestions are supported by carefully controlled experimental evi- 
dence, they must be subjected to examination before they are used as 
working hypotheses in experimental work. 

The first suggestion, that the fungus is carried with the seed, de- 
serves careful attention. This might happen in three ways. Spores 
might adhere to the seed coat, spores or fragments of the fungus 
might accompany the seed, and living mycelium of the fungus might 
occur within the seed. When the conditions under which seed is 
produced are examined it is found that the fungus spores are prac- 
tically all discharged and blown away before the seed is thrashed, 
thus making it highly improbable that spores are attached to the seed 
except as a rare occurrence. Commercial seed is so well cleaned that 
there appears to be small chance that fragments of the fungus are 
often conveyed with the seed. 

The possibility that fragments of the fungus as well as the spores 
may be carried with the seed appears unlikely in the case of most 
commercial seed. Debris consisting of plant parts is so light in com- 
j:>arison with the seed that it is easily removed. Nevertheless, Ivy 
Massee (1914) states that she has examined commercial seed and 

1 On April G, 1919, apothecia of Psrudope.iza trifolii were found abundantly on living 
overwintered clover loaves at Madison, Wis., showing clearly that young leaves infected 
late in the autumn under favorable conditions may carry the fungus over winter. 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 31 

found the fungus present. This can hardly be a common occurrence 
in America. 

The possibility that the fungus may be present in the seed as 
mycelium is open to the objection that this involves a larger or 
smaller amount of systemic infection, both of the plant producing 
the seed and of the seedling. No evidence of such a relation of these 
fungi to their host plants has been found, and therefore this method 
appears highly improbable. 

The second suggestion, that the fungus is conveyed with plant 
debris that accompanies soil which is transported to new fields, may 
and probably does account for a small amount of the distribution of 
the fungus. But owing to the restricted extent of this practice, this 
method must be of minor importance. 

The third hypothesis, that spores of Pseudopeziza are generally 
distributed in the air, has been advanced several times in a vague 
way either in a discussion of conditions where large areas of dis- 
eased alfalfa were growing at no great distance or with an implied 
belief that some other host plant in the vicinity was the source from 
which this general infection arose. In the vicinity of areas of dis- 
eased alfalfa it is highly probable that spores are borne to a consid- 
erable distance by wind, but it is not often that the spores are pro- 
duced so abundantly that they are likely to be conveyed great 
distances in large numbers. 

The final hypothesis, that other hosts provide the source of infec- 
tion, has been rendered less probable by results already presented. 
The only common hosts that can be considered are red clover and yel- 
low trefoil (Medicago lupulina). Pseudopeziza on red clover appears 
to be a distinct species from that on alfalfa, and no evidence has 
been obtained indicating that the fungus on yellow trefoil can cross 
to that host. 

Thus, a summary of the available evidence does not point clearly 
to any of these suggestions as the one most likely to contain the 
truth. However, the suggestion that the fungus is carried with the 
seed affords most opportunity for experimental study and, if found 
true, affords the greatest opportunity for the application of control 
measures. ' The following experiments in seed sterilization were car- 
ried out. 

EXPERIMENTAL METHODS AND RESULTS. 

Laboratory experiments. — If the fungus spores are carried adherent 
to the seed they must inevitably germinate upon the seed coat and 
produce apothecia there, if at all. In order to determine to what 
extent the fungus is capable of developing upon the seed coat, spores 
were discharged upon seeds sterilized with formaldehyde. The seeds 
were then germinated upon agar in test tubes. After a time minute 



32 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 

fungous colonies were found developing upon the seed coats, whether 
they remained attached to the cotyledon leaves or fell to the agar. 
In no case were the cot3dedon leaves attacked. Subsequent attempts 
to infect cotyledon leaves of seedlings did not produce macroscopic 
lesions. The seed coats bearing the minute fungous colonies were 
subsequently transferred to fresh agar slopes to keep them moist, and 
finally they developed minute apothecia. This development was so 
slow, however, that it is doubtful whether it could proceed so far 
under field conditions, where periods of drying would occur and com- 
petition with other fungi would be encountered. 

Efforts to grow the fungus on soil sterilized or unsterilized have 
been entirely unsuccessful. 

If the fungus occurs on the outside of the seed or in debris it can 
easily be destroyed by the surface sterilization of the seed. If such 
treated seed can then be grown under conditions which will exclude 
other sources of infection and which will also be favorable for the 
development of the fungus, the occurrence of the disease Mall indi- 
cate that the fungus is carried within the seed. 

A satisfactory method of seed sterilization has been worked out 
by Mr. A. H. Gilbert (in an unpublished manuscript). He found 
that treatment with a solution of 1 part of bichlorid of mercury in 
1,000 parts of water for five minutes rendered the seed sterile, while 
treatment for 10 minutes injured the seed. These treatments were 
repeated, and it was found that treatment for eight minutes was 
more than sufficient to render the seeds sterile without injury, pro- 
vided they were washed promptly after treatment. All sterilized 
seed mentioned in the experiments here described were treated in 
this way. 

Suitable conditions for growing the treated seed Avere difficult to 
obtain. Two places were tried — in the greenhouse during the winter 
months and in the open field in localities as remote as possible from 
other alfalfa. Experience in the greenhouse in the winter of 1915 
showed that unless great care was taken with infected plants, the 
fungus was likely to occur occasionally on other alfalfa plants in the 
same house. During the following two winters all inoculated plants 
were cared for so thoroughly that in not a single instance did the leaf- 
spot develop upon any other plant in the houses until the disease 
appeared in the fields outside in the spring. Alfalfa plants grown 
close to red clover infested with Pseudopeziza trifolii remained free 
from leaf-spot. The following greenhouse-plat trials were made : 

(1) Pour grains of sterilized alfalfa seed were exposed to a discharge of 
ascospores of Pseudopeziza medicaffinis for three days before sowing in the 
garden greenhouse on March 3, 1916. Thousands of viable spores must have 
been attached to the seeds at the time they were sown. By April 15 the plants 
were 6 inches tall and very vigorous. When the experiment was discontinued 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 33 

on May 10 no trace of Pseudopeziza had appeared. The plants were then 10 
to 12 inches tall, very vigorous, and in a dense mat apparently favorable for 
the development of the fungus. At the end of this experiment the plat was 
inoculated with Pseudopeziza from overwintered leaves and was quickly over- 
run with the disease, showing that the greenhouse conditions were favorable 
for its development. 

(2) On January 27, 1917, a plat about 4 feet square was sown in the garden 
greenhouse with unsterilized Kansas-grown alfalfa seed. This plat developed 
normally without leaf-spot until May 5, when the disease was present in the 
field outside. 

(3) On February 16, 1917, a plat 2 by 3 feet was sown in a garden green- 
house with sterilized alfalfa seed 3 years old. This plat likewise developed 
normally with no leaf-spot until May 10, at which date leaf-spot was abundant 
outside the greenhouse. 

Several other plats were started and developed in the green- 
house without leaf-spot, like those referred to above, but owing to 
insect injury the conditions were not as favorable for the develop- 
ment of the disease as those described. In fact, all greenhouse plats 
started from seed, whether sterilized, unsterilized, or even treated 
with spores before sowing, have developed without tlie appearance 
of leaf-spot until the disease occurred abundantly in an alfalfa 
field close outside the greenhouse. 

Field plats. — In the selection of locations for plats three conditions 
were sought: (1) Remoteness from large areas of growing alfalfa; 
(2) the greatest possible distance from farms where alfalfa has been 
grown; and (3) accessibility, so that a visit to the plat would be 
possible. The second condition was very difficult to secure. Small 
plats of alfalfa are surprisingly abundant even in localities where it is 
not grown as a farm crop. In consequence of this fact, only one of 
the eight plats started in 1915 was found upon examination to be 
sufficiently remote to give results of value. 

In 1915 the assistance of the States Relations Service secured the 
cooperation of several agricultural county agents whose intimate 
knowledge of local conditions made possible the selection of a larger 
number of suitable locations. To these men the writer is indebted 
for any degree of success that was attained in these experiments. 

The seed which had been sterilized superficially was furnished to 
the agricultural county agents, who allotted it to the men on whose 
farms the plats were to be located. In the autumn all the plats 
were visited except the one at Bruce, S. Dak., which was under the 
observation of Dr. A. G. Johnson, and the presence or absence of 
leaf-spot was determined. In a number of cases alfalfa was found 
growing nearer the plat than was previously supposed. The results 
noted on such plats — always an abundance of leaf-spot — are excluded 
from the summary in Table VIII. However, if, as sometimes oc- 
curred, the near-by plants were very few in number and no other 



34 



BULLETIN 759, U. S. DEPARTMENT OE AGRICULTURE. 



plants were known to exist within a 5-mile radius the results have 
been included. 



Table VIII. — Summary of data of plat tests to determine whether leaf-spot can 
he prevented on alfalfa sown in isolated localities by the superficial steriliza- 
tion of alfalfa seed. 



Stale and (own. 



Date 
sown. 



Date 
visited. 



Area 
(square 
rods). 



Distance 

to nearest 

alfalfa 

(miles). 



Leaf-spot. 



Maine: 
Mercer. 



Albion 

Fairfield 

Gorham 

Harrison 

Vassalboro.. 

Windsor 

South Dakota: 

Bruce 

Wisconsin: 

Doering 

McConnor . . 

Radisson . . . 

Merrill 

Do 

Do 

Tomahawk. 
Do 

Weirgor 



1915. 

June 28 

1916. 
May 6 

June 

May 20 
June 1 

May 

June 

May 20 



June. 
May.. 
June. 
..do. 
..do.. 
..do., 
..do. 
..do. 
..do.. 



1916. 
June 14 

Sept. 12 
...do.... 
Sept. 5 
...do.... 
Sept. 6 
Sept. 12 

Aug. 11 



Oct. 

Oct. 

...do. 

Oct. 

...do. 

...do. 

...do. 

...do. 

Oct. 



3£ 
5 

i 
3| 
5 
5 

5 

10 
i 

i 

5 

8 

12', 
10 
10 

5 



Present. 

Do. 
Do. 
Do. 

Doubtful. 
Present. 
Do. 

Do. 

Doubtful. 
Present. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



From Table VIII it appears that the one small well-isolated plat 
started in 1015 did not develop leaf-spot until the following year. 
Of the 16 plats started in 1916 only 2 failed to show an abundance 
of leaf-spot in the autumn of the same year. One of these plats, 
located at Harrison, Me., was in very poor condition, only a few 
scattering spindling plants about 4 inches high being found. How- 
ever, other plats in almost as poor condition showed leaf -spot. The 
second doubtful plat, at Doering, Wis., was in very vigorous con- 
dition, but it had been cut just previous to inspection, leaving very 
little foliage. Unfortunately, it was not feasible to .revisit the plat 
the following year. 

These results are in accord with previous experiments and experi- 
ence. Surface sterilization of seed apparently accomplishes nothing 
in excluding leaf-spot from alfalfa fields. But these results do point 
very clearly to one conclusion, that the leaf-spot fungus is not car- 
ried on or in debris mixed with the seed. The greenhouse experi- 
ments, which are only suggestive because of their limited extent, 
indicate that the fungus is not carried within the seed. 

Thus, in conclusion, it is necessary to say that no positive evidence 
pointing toward the method by which this disease gains access to 
remote alfalfa fields has been found. Evidence has been obtained 
which apparently eliminates other plants previously under suspicion 
as host plants of the fungus from consideration as sources. A lim- 



LEAF-SPOT OF ALFALFA AND RED CLOVER. 35 

ited amount of evidence indicates that the fungus is not carried 
with the seed. Yet the fact of the almost universal occurrence of the 
disease in remote localities is well established. The explanation of 
this fact still furnishes a very interesting and apparently very 
difficult problem. 

SUMMARY. 

( 1 ) One of the most important diseases, if not the most important 
foliage disease, of alfalfa is the leaf-spot caused by the fungus 
Pseudopeziza medicaginis (Lib.) Sacc. A similar but less important 
leaf-spot of red clover is caused by the fungus Pseudopeziza trlfolii 
(Biv.-Bern.) Fckl. The morphological differences between these 
fungi are so slight that doubt has frequently been expressed whether 
they are not identical. Several conflicting opinions as to the life 
histories of these fungi are found in mycological literature. This 
study attempts to determine the relationship of the two fungi here 
mentioned and to trace as far as possible their life histories in rela- 
tion to their host plants. 

(2) Pseudopeziza medicaginis on alfalfa and Pseudopeziza trifolii 
on red clover have been obtained and studied in pure culture. Efforts 
to cross these fungi from one host to the other have not been success- 
ful. Morphological as well as physiological differences have been 
found which in the opinion of the writer justify retaining the fungi 
as distinct species. 

(3) None of the imperfect fungi which have been regarded as a 
stage in the development of these fungi have been found to be re- 
lated. Apparently no other spore form than the ascospore occurs 
in nature. 

(4) Infection is produced by the direct penetration of the germi- 
nating ascopores through the cuticle and epidermal cell wall of the 
leaf. The mycelium developing into a small stroma about the point 
of entry produces in about two weeks an apothecium. 

(5) The fungus lives over winter on dead leaves which escape 
decay, and ascopores produced in the spring furnish the source of 
new infection. 

(6) Efforts to exclude the disease from alfalfa fields sown in 
localities remote from other alfalfa by the surface sterilization of 
the seed have given no degree of success. Evidently, in these experi- 
ments at least, the fungus was not carried on the surface of the seed — 
probably not with the seed at all. The demonstration of the source 
of infection in such fields still furnishes an interesting problem. 



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36 



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